Chimeric antigen receptor cells specifically targeting tumors, their preparation methods and applications

Abstract

This invention relates to the field of biotechnology, specifically to a chimeric antigen receptor specifically targeting tumors, its preparation method, and its applications, including polynucleotides, expression vectors, recombinant viruses, recombinant cells, compositions, and pharmaceutical uses. The chimeric antigen receptor specifically targeting and binding to HLA-G provided by this invention has an extracellular domain consisting of antigen-binding domains of ILT2 and ILT4 receptors. Compared to CAR-modified immune cells whose extracellular domains are antibody antigen-binding regions, the HLA-G-targeting immune response cells provided by this invention can broadly recognize numerous tumor cells, greatly improving the killing efficiency and precision against various types of tumor cells. Furthermore, the extracellular domain, consisting of antigen-binding domains of ILT2 and ILT4 receptors, is the binding domain of natural receptors, making it safer and more reliable than artificially synthesized antibodies, and accurately reflecting the target binding ability in its natural state.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a chimeric antigen receptor that specifically targets tumors, its preparation method and application, including polynucleotides, expression vectors, recombinant viruses, recombinant cells, compositions, and pharmaceutical uses. Background Technology

[0002] With the rapid development of biotechnology, immunotherapy has become one of the main treatments for cancer. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, antibody therapy, and immune checkpoint blockade therapy. Among these, in the field of adoptive cell therapy, chimeric antigen receptor-modified immunocellular therapy, especially chimeric antigen receptor-modified T-cell (CAR-T) therapy, is currently very popular and is a star therapy in this field.

[0003] CAR-T cell immunotherapy works by modifying a patient's own T cells with chimeric antigen receptors using genetic engineering techniques. These modified chimeric receptors allow CAR-T cells to specifically recognize tumor-associated antigens (tumor cell markers), thereby targeting and killing the tumor. Compared to ordinary immune cells, CAR-T cells exhibit higher targeting specificity, killing activity, and persistence. Currently, modified immunotherapy, represented by CAR-T cells targeting CD19 and BCMA, has shown significant efficacy in treating hematologic malignancies such as lymphoma and multiple myeloma, and is considered one of the most promising cancer treatment methods.

[0004] However, since over 90% of cancer patients suffer from solid tumors, the types of solid tumors and the number of tumor-specific target antigens on their surfaces require further identification. One of the biggest challenges in applying CAR-T immunotherapy to solid tumor treatment lies in the extremely high specificity required of CAR-T cells for antigen expression on tumor cells. Otherwise, it can easily lead to continuous T cell activation that kills normal cells or releases large amounts of cytokines, causing serious side effects. Although CAR-T cell immunotherapy requires a very high specificity for tumor cell antigen expression, the selectivity of tumor-specific target antigens is limited, and most antigens expressed by tumors lack tumor specificity. CAR-T cell immunotherapy targeting tumor-associated antigens suffers from problems such as off-target effects and drug resistance.

[0005] Therefore, finding broader yet more specific, more efficient, and safer molecules as targets for CAR therapy is an urgent problem to be solved in chimeric antigen receptor modified immunocellular therapy. The key to finding these targets lies in the fact that the target antigen is highly expressed on the surface of tumor cells, but not expressed or poorly expressed on the surface of normal cells. Summary of the Invention

[0006] Human leukocyte antigen G (HLA-G) is a non-classical HLA-I class molecule. HLA-G can be cleaved into seven isoforms, including membrane-bound HLA-G1, HLA-G2, HLA-G3, and HLA-G4, and soluble HLA-G5, HLA-G6, and HLA-G7. Of these seven isoforms, two are recognized by the ILT2 receptor and five by the ILT4 receptor. HLA-G molecules are widely expressed on the surface of numerous tumor cells (especially solid tumors). By interacting with the ILT2 receptor on the surface of immune cells, it activates downstream pathways of the immune system, transmitting inhibitory signals to the immune cells and leading to immunosuppression to escape the attack of the immune system. Therefore, it is considered an important immune checkpoint molecule. Furthermore, HLA-G molecules are rarely expressed or only transiently expressed in healthy tissues, representing a tumor cell-specific alteration during tumorigenesis. This makes it a precise target for immunotherapy of tumors, especially solid tumors, providing insights for the development of related new therapies and drugs.

[0007] CARs typically consist of an extracellular targeting domain, a transmembrane spacer, and an intracellular signaling domain. The targeting domain of a CAR usually originates from the antigen-binding region of an antibody, specifically the heavy and light chains of immunoglobulins that form the antibody binding site. A common example is the single-chain variable fragment scFv of a monoclonal antibody responsible for recognizing and binding antigens. CAR-modified immune cells with the antibody's antigen-binding region as the extracellular domain have advantages such as high specificity and sensitivity. However, they also present the following problems: due to the high specificity of the antibody, one antibody can usually only bind to one target, easily leading to "off-target" events when targeting tumor cells. For most tumors, the available target molecules for CAR immunotherapy are scarce, and no target molecule is suitable for broad-spectrum tumor treatment.

[0008] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technologies.

[0009] Therefore, a first aspect of the present invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, the chimeric antigen receptor comprising:

[0010] The extracellular domain includes at least one binding domain for ILT2 targeting HLA-G and at least one binding domain for ILT4 targeting HLA-G.

[0011] A transmembrane domain, which is connected to the extracellular domain;

[0012] Intracellular domain, which is connected to the transmembrane domain.

[0013] The chimeric antigen receptor specifically targeting HLA-G provided by this invention comprises an extracellular domain including the antigen-binding domain of the ILT2 receptor and the antigen-binding domain of the ILT4 receptor. Compared to CAR-modified immune cells in the prior art, which use the antigen-binding region of the antibody as the extracellular domain, the engineered HLA-G-targeting immune response cells of this invention can broadly recognize a wide range of tumor cells, such as solid tumor cells that are positive for any one of HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7 on their cell surface, greatly improving the killing efficiency and precision against various types of tumor cells. Furthermore, the extracellular domains, including the antigen-binding domains of the ILT2 and ILT4 receptors, are the binding domains of the natural receptors, making them safer and more reliable than artificially synthesized antibodies, and accurately reflecting the target binding ability in the natural state. Therefore, this invention provides a promising new approach for tumor treatment.

[0014] According to a specific embodiment of the present invention, the binding domain of ILT2 that targets HLA-G has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity with it.

[0015] The binding domain of ILT4 that targets HLA-G has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence that is at least 80% identical to it.

[0016] According to a specific embodiment of the present invention, the binding domain of ILT2 that targets HLA-G and the binding domain of ILT4 that targets HLA-G are connected by a linker peptide.

[0017] According to a specific embodiment of the present invention, the linker peptide includes any one of a flexible linker, a rigid linker, and a shearable linker.

[0018] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, which is connected to the binding domain of ILT2 targeting HLA-G and the binding domain of ILT4 targeting HLA-G.

[0019] According to a specific embodiment of the present invention, the signal peptide includes at least one derived from CD8α signal peptide, IgG signal peptide, and CD28 signal peptide.

[0020] According to a specific embodiment of the present invention, the signal peptide is derived from the CD8α signal peptide.

[0021] According to a specific embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 7.

[0022] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0023] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region, or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region, or at least a portion of an immunoglobulin, modified by one or more amino acids.

[0024] According to a specific embodiment of the present invention, the hinge domain originates from the CD8α hinge region.

[0025] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80% identity with it.

[0026] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the following: CD8α transmembrane structural region, CD28 transmembrane structural region, CD3ζ transmembrane structural region, CD4 transmembrane structural region, 4-1BB transmembrane structural region, OX40 transmembrane structural region, ICOS transmembrane structural region, CTLA-4 transmembrane structural region, PD-1 transmembrane structural region, LAG-3 transmembrane structural region, 2B4 transmembrane structural region, BTLA transmembrane structural region, and synthetic peptide, wherein the synthetic peptide does not contain proteins related to immune response.

[0027] According to a specific embodiment of the present invention, the transmembrane domain originates from the CD8α transmembrane structural region.

[0028] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 9.

[0029] According to a specific embodiment of the present invention, the intracellular domain includes an immune receptor tyrosine activation motif and a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain and the immune receptor tyrosine activation motif are linked by a linker peptide.

[0030] According to a specific embodiment of the present invention, since HLA-G usually transmits inhibitory signals to immune cells after binding to them, thereby inhibiting the function of immune cells, the extracellular segment of the chimeric antigen receptor designed in this invention is the extracellular region of the inhibitory receptors ILT2 and ILT4 that recognize HLA-G. By replacing the intracellular segment of these inhibitory receptors with a co-stimulatory signal domain, the signal recognizing HLA-G can be converted into an activation signal to activate immune cells. The CAR-modified immune cells prepared in this invention can resist the inhibition from the tumor immune microenvironment and reverse immune cell depletion.

[0031] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif originates from the intracellular signal transduction domain of the CD3ζ chain or the intracellular signal transduction domain of FcεRIγ.

[0032] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif originates from the intracellular signal transduction domain of the CD3ζ chain.

[0033] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif has an amino acid sequence as shown in SEQ ID NO: 11.

[0034] According to a specific embodiment of the present invention, the co-stimulatory signal domain includes at least one of the following: CD28 intracellular signal transduction domain, 4-1BB intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD40 or CD40L intracellular signal transduction domain, and ICOS intracellular signal transduction domain.

[0035] According to a specific embodiment of the present invention, the co-stimulatory signal domain originates from the 4-1BB intracellular signal transduction domain.

[0036] According to a specific embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 10.

[0037] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22.

[0038] A second aspect of the present invention provides an isolated polynucleotide encoding the chimeric antigen receptor described in the first aspect.

[0039] A third aspect of the present invention provides an expression vector carrying the polynucleotide described in the second aspect.

[0040] A fourth aspect of the present invention provides a recombinant virus carrying the polynucleotide described in the second aspect or the expression vector described in the third aspect, wherein the recombinant virus is capable of expressing the chimeric antigen receptor described in the first aspect.

[0041] The fifth aspect of the present invention provides a recombinant cell carrying the polynucleotide described in the second aspect or the expression vector described in the third aspect, or infected with the recombinant virus described in the fourth aspect, capable of expressing the chimeric antigen receptor described in the first aspect.

[0042] According to a specific embodiment of the present invention, the recombinant cells are derived from immune cells.

[0043] According to a specific embodiment of the present invention, the immune cells are obtained by isolating from peripheral blood, umbilical cord blood, or tissues and organs, or the immune cells are obtained by differentiating from stem cells or immune cell progenitor cells.

[0044] According to a specific embodiment of the present invention, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells that have not undergone in vivo development and are within 14 days of fertilization.

[0045] According to a specific embodiment of the present invention, the immune cells include at least one selected from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

[0046] A sixth aspect of the present invention provides a composition comprising at least one of the following:

[0047] The chimeric antigen receptor described in the first aspect;

[0048] The polynucleotides described in the second aspect;

[0049] The expression carrier described in the third aspect;

[0050] The recombinant virus described in the fourth aspect;

[0051] The recombinant cells described in the fifth aspect.

[0052] The seventh aspect of the present invention provides the use of the composition described in the sixth aspect in the preparation of an antitumor medicament.

[0053] According to a specific embodiment of the present invention, the tumor cells express HLA-G.

[0054] To overcome the problem of the scarcity of effective targets for most solid tumors in existing cell therapy clinical applications, this invention provides a chimeric antigen receptor targeting human HLA-G, its gene and recombinant expression vector, engineered HLA-G-targeting chimeric antigen receptor-modified immune response cells, and their applications.

[0055] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0056] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0057] Figure 1 The results of the verification of ILT receptor expression in CAR-NK cells in Example 3 of the present invention are shown;

[0058] Figure 2 The screening results of HLA-G positive tumor cell lines in Example 4 of the present invention are shown;

[0059] Figure 3 The results of CAR-NK cell killing of K562 cells in Example 5 of this invention are shown;

[0060] Figure 4 The results of CAR-NK cell killing NCI-H716 cells in Example 5 of this invention are shown.

[0061] Figure 5 The results of CAR-NK cells killing SKOV3 cells in Example 5 of this invention are shown.

[0062] Figure 6 The results of CAR-NK cell killing of Huh7 cells in Example 5 of this invention are shown;

[0063] Figure 7 The results of CAR-NK cell therapy for mouse hematologic malignancies in Example 6 of this invention are shown.

[0064] Figure 8 The results of CAR-NK cell therapy on mouse ovarian cancer subcutaneous xenografts in Example 6 of this invention are shown, wherein... Figure 8 A in the figure shows the tumor proliferation curves for each experimental group. Figure 8 B in the figure shows the statistical results of tumor weight in each experimental group after the experiment was terminated. Figure 8 C in the figure shows images of tumors in each experimental group after the experiment was terminated;

[0065] Figure 9 The results of CAR-NK cell therapy on mouse subcutaneous colon cancer xenografts in Example 6 of this invention are shown, wherein... Figure 9 A in the figure shows the tumor proliferation curves for each experimental group. Figure 9 B in the figure shows the statistical results of tumor weight in each experimental group after the experiment was terminated. Figure 9 C in the figure shows images of tumors in each experimental group after the experiment was terminated. Detailed Implementation

[0066] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0067] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0068] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0069] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0070] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0071] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0072] In this invention, the term "effect-to-target ratio" refers to the ratio of effector cells (specific CAR-NK cells targeting HLA-G) to target cells (tumor cells).

[0073] In this invention, the term "chimeric antigen receptor" is abbreviated as CAR. It is an artificial receptor molecule manufactured using genetic engineering technology, comprising an antigen-binding domain (usually derived from a single-chain antibody variable region gene fragment, scFV; or derived from a single-domain antibody), a hinge domain, a transmembrane domain, and an intracellular signal transduction domain.

[0074] In this invention, the term "reduced expression" refers to the elimination or reduction of the expression of one or more gene products encoded by a gene in a cell compared to the normal expression level of the gene's mRNA or protein. In some cases, this effect on gene expression is temporary or reversible, such as by inhibiting gene expression through small molecule compounds, nucleic acids such as RNAi, siRNA, and shRNA, or specific proteins. In other cases, this effect on gene expression is permanent, such as by introducing base insertions or deletions into the coding region of the target gene through gene editing, causing frameshift mutations, or by inserting a DNA sequence into the coding or non-coding region of the target gene through homologous recombination, causing frameshift mutations, premature termination of transcription and / or translation, or translation into a completely different polypeptide, thus permanently inactivating the gene. The percentage of reduced gene expression caused by different methods can be 100%, 90% or more, 80% or more, 70% or more, 60% or more, 50% or more, 40% or more, 30% or more, or 20% or more.

[0075] In this invention, the term "hinge region" refers to the region between the CH1 and CH2 regions of the antibody heavy chain. This region includes interchain disulfide bonds, is rich in proline, does not form α-helices, and is prone to stretching and some degree of twisting, which facilitates complementary binding between the antibody's antigen-binding site and the antigen epitope. In the heavy chain single-domain antibody of this invention, the variable region of the heavy chain is connected to the Fc region through the hinge region.

[0076] In this invention, the term "cell" refers to a eukaryotic cell, particularly a mammalian cell, such as a human cell.

[0077] In this invention, the term "NK cell" refers to natural killer cells, which are generally CD56+CD45+ double positive and express multiple activation receptors, such as NKG2D, NKp44, and NKp46. NK cells can differentiate from pluripotent stem cells (such as iPSCs), be isolated from peripheral blood, differentiate from CD34+ cells (such as umbilical cord blood or placental blood), or be obtained from tumor cell lines (such as NK92).

[0078] In this invention, the term "signal peptide," also called a signal sequence, refers to a localization tag for membrane proteins, secretory proteins, and lysosomal proteins. It is typically located at the amino terminus of a protein and consists of 13-26 residues. Signal peptides do not have a conserved amino acid sequence, but they do possess conserved structural features.

[0079] As used herein, the term "immune cell" refers to cells derived from humans. More specifically, the immune cells of this invention can be derived from pluripotent stem cells through directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages. The immune cells of this invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be isolated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages. The immune cells of this invention can also be isolated from human blood, bone marrow, lymph nodes or lymphoid organs, or specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs, and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, such as bone marrow or lymphocytes, typically T cells, NK cells, macrophages, etc. Preferably, according to this invention, the immune cells include T cells, NK cells, and macrophages. The immune cells of this invention can also be derived from immune cell progenitor cells, such as lymphocyte progenitor cells.

[0080] In this invention, the term "pluripotent stem cell" generally refers to human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESCs are obtained from blastocysts during the development of human fertilized eggs, or from embryos obtained through nuclear transfer (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development); human iPSCs can be obtained from human cells through reprogramming by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, MYC). Both human ESCs and iPSCs express OCT4, NANOG, Tra-1-60, and SSEA-4, while those that do not express SSEA-1. A significant characteristic of pluripotent stem cells is their ability to differentiate into three germ layer tissues or cells in vitro or in vivo.

[0081] In this invention, the term "hematopoietic stem cell" refers to CD34+ cells. These cells can be isolated from peripheral blood, umbilical cord blood, placental blood, bone marrow, spleen, lymph, or other tissues, or differentiated from pluripotent stem cells.

[0082] In this invention, the term "progenitor cell" refers to a cell that can differentiate into various types of immune cells, such as myeloid stem cells, lymphoid stem cells, and further, erythroid stem cells, granulocytic and monocyte stem cells, megakaryocyte stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0083] In this invention, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vector (e.g., expression vector) provided by the present invention contains a nucleic acid sequence encoding a fusion protein as described in the present invention, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0084] In this invention, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0085] Chimeric antigen receptor

[0086] This invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, the chimeric antigen receptor comprising:

[0087] The extracellular domain includes at least one binding domain for ILT2 targeting HLA-G and at least one binding domain for ILT4 targeting HLA-G.

[0088] A transmembrane domain, which is connected to the extracellular domain;

[0089] Intracellular domain, which is connected to the transmembrane domain.

[0090] ILT2 and ILT4 receptors are two receptors on the surface of immune cells responsible for binding to HLA-G molecules. ILT4 is expressed only on myeloid-derived immune cells, while ILT2 is expressed on both lymphoid and myeloid-derived immune cells. Both ILT2 and ILT4 receptors are divided into extracellular, transmembrane, and intracellular regions, with the extracellular region being the functional area responsible for recognizing and binding HLA-G. The extracellular region of the ILT2 receptor is further divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4). According to reports, domains D1 and D2 are responsible for binding to HLA-G, while domains D3 and D4 only support the external structure formed after D1 and D2 bind to HLA-G.

[0091] In one specific embodiment of the present invention, this application provides a synthetic polypeptide that targets and binds to HLA-G, wherein the synthetic polypeptide sequence is derived from a combination of polypeptide sequences derived from ILT2 and ILT4. In general, the synthetic polypeptide is formed by linking polypeptide sequences derived individually from ILT2 or ILT4 together, including but not limited to the following compositions: ILT2-D1D2+Linker+ILT4-D1D2, ILT4-D1D2+Linker+ILT2-D1D2, ILT2-ECD+Linker+ILT4-ECD, ILT4-ECD+Linker+ILT2-ECD, ILT2-D1D2+Linker+ILT4-ECD, ILT4-D1D2+Linker+ILT2-ECD, ILT2-D1+Linker+ILT4-ECD, ILT4-D1D2+Linker+ILT2-ECD, ILT2-D1+Linker+ILT4-ECD, ILT4-D1D2+Linker+ILT2-ECD, ILT2-D1+Linker+Linker+ILT2-ECD, ILT2-D1+Linker+Linker+ILT4 ... r+ILT4-D1, ILT2-D1+Linker+ILT4-D2, ILT2-D1+Linker+ILT4-D1D2, ILT2-D1+Linker+ILT4-ECD, ILT2-D1+Linker+ILT2-D1, ILT2-D1+Lin ker+ILT2-D2, ILT2-D1+Linker+ILT2-D1D2, ILT2-D1+Linker+ILT2-ECD, ILT4-D1+Linker+ILT4-D1, ILT4-D1+Linker+ILT4-D2, ILT4-D1+Li nker+ILT4-D1D2, ILT4-D1+Linker+ILT4-ECD, ILT4-D1+Linker+ILT2-D1, ILT4-D1+Linker+ILT2-D2, ILT4-D1+Linker+ILT2-D1D2, ILT4-D 1+Linker+ILT2-ECD, ILT2-D2+Linker+ILT4-D1, ILT2-D2+Linker+ILT4-D2, ILT2-D2+Linker+ILT4-D1D2, ILT2-D2+Linker+ILT4-ECD, ILT 2-D2+Linker+ILT2-D1, ILT2-D2+Linker+ILT2-D2, ILT2-D2+Linker+ILT2-D1D2, ILT2-D2+Linker+ILT2-ECD, ILT4-D2+Linker+ILT4-D1, I LT4-D2+Linker+ILT4-D2, ILT4-D2+Linker+ILT4-D1D2, ILT4-D2+Linker+ILT4-ECD, ILT4-D2+Linker+ILT2-D1, ILT4-D2+Linker+ILT2-D2,ILT4-D2 + Linker + ILT2-D1D2, ILT4-D2 + Linker + ILT2-ECD, ILT2-D1D2 + Linker + ILT4-D1, ILT2-D1D2 + Linker + ILT4-D2, ILT2-D1D2 + Linker + ILT4-D1D2, ILT2-D1D2 + Linker + ILT4-ECD, ILT2-D1D2 + Linker + ILT2-D1, ILT2-D1D2 + Linker + ILT2-D2, ILT2-D1D2 + Linker + ILT2-D1D2, ILT2-D1D2 + Linker + ILT2-ECD, ILT4-D1D2 + Linker + ILT4-D1, ILT4-D1D2 + Linker + ILT4-D2, ILT4-D1D2 + Linker + ILT4-D1D2, ILT4-D1D2 + Linker + ILT4-ECD, ILT4-D1D2 + Linker + ILT2-D1, ILT4-D1D2 + Linker + ILT2-D2, ILT4-D1D2 + Linker + ILT2-D1D2, ILT4-D1D2 + Linker + ILT2-ECD, ILT2-ECD + Linker + ILT4-D1, ILT2-ECD + Linker + ILT4-D2, ILT2-ECD + Linker + ILT4-D1D2, ILT2-ECD + Linker + ILT4-ECD, ILT2-ECD + Linker + ILT2-D1, ILT2-ECD + Linker + ILT2-D2, ILT2-ECD + Linker + ILT2-D1D2, ILT2-ECD + Linker + ILT2-ECD, ILT4-ECD + Linker + ILT4-D1, ILT4-ECD + Linker + ILT4-D2, ILT4-ECD + Linker + ILT4-D1D2, ILT4-ECD + Linker + ILT4-ECD, ILT4-ECD + Linker + ILT2-D1, ILT4-ECD + Linker + ILT2-D2, ILT4-ECD + Linker + ILT2-D1D2, ILT4-ECD + Linker + ILT2-ECD, preferably: ILT2-D1D2 + Linker + ILT4-D1D2,ILT4-D1D2+Linker+ILT2-D1D2, wherein the Linker is a common linker peptide sequence, such as GGGGS (SEQ ID NO: 25). It should be noted that any linker peptide known in the art capable of linking two proteins without affecting protein function is applicable to this invention. The linker peptide includes any one of flexible Linkers, rigid Linkers, and shearable Linkers.

[0092] According to a specific embodiment of the present invention, the binding domain of ILT2 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity with it.

[0093] The binding domain of ILT4 that targets HLA-G has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence that is at least 80% identical to it.

[0094] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, which is connected to the binding domain of ILT2 targeting HLA-G and the binding domain of ILT4 targeting HLA-G.

[0095] According to a specific embodiment of the present invention, the signal peptide includes at least one derived from CD8α signal peptide, IgG signal peptide, and CD28 signal peptide.

[0096] According to a specific embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 7.

[0097] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0098] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region, or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region, or at least a portion of an immunoglobulin hinge domain modified by one or more amino acids.

[0099] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 80% identity with it.

[0100] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the following: CD8α transmembrane structural region, CD28 transmembrane structural region, CD3ζ transmembrane structural region, CD4 transmembrane structural region, 4-1BB transmembrane structural region, OX40 transmembrane structural region, ICOS transmembrane structural region, CTLA-4 transmembrane structural region, PD-1 transmembrane structural region, LAG-3 transmembrane structural region, 2B4 transmembrane structural region, BTLA transmembrane structural region, and synthetic peptide, wherein the synthetic peptide does not contain proteins related to immune response.

[0101] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO: 9.

[0102] According to a specific embodiment of the present invention, the intracellular domain includes an immune receptor tyrosine activation motif and a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain and the immune receptor tyrosine activation motif are linked by a linker peptide.

[0103] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif originates from the intracellular signal transduction domain of the CD3ζ chain or the intracellular signal transduction domain of FcεRIγ.

[0104] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif originates from the intracellular signal transduction domain of the CD3ζ chain.

[0105] According to a specific embodiment of the present invention, the immune receptor tyrosine activation motif has an amino acid sequence as shown in SEQ ID NO: 11.

[0106] According to a specific embodiment of the present invention, the co-stimulatory signal domain includes at least one of the following: CD28 intracellular signal transduction domain, 4-1BB intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD40 or CD40L intracellular signal transduction domain, and ICOS intracellular signal transduction domain.

[0107] According to a specific embodiment of the present invention, the co-stimulatory signal domain originates from the 4-1BB intracellular signal transduction domain.

[0108] According to a specific embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 10.

[0109] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 22.

[0110] According to an embodiment of the present invention, from the N-terminus to the C-terminus, the CAR may sequentially comprise a synthetic polypeptide derived from ILT2 and ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signal transduction domain.

[0111] According to a specific embodiment of the present invention, from the N-terminus to the C-terminus, the CAR may sequentially include a synthetic polypeptide that binds HLA-G derived from ILT2 and ILT4, a hinge region derived from CD8α, a transmembrane domain derived from CD8α, a co-stimulatory signaling domain derived from 4-1BB, and an intracellular signal transduction domain derived from CD3ζ.

[0112] According to a specific embodiment of the present invention, from the N-terminus to the C-terminus, the CAR may sequentially include a signal peptide, a synthetic polypeptide derived from ILT2 and ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signal transduction domain.

[0113] According to a more specific embodiment of the present invention, the inventors designed two synthetic polypeptides that can target and bind to human HLA-G, as shown in Table 1. Both synthetic polypeptides are composed of polypeptide sequences derived from ILT2 and ILT4. The synthetic polypeptides derived from ILT2 and ILT4 have amino acid sequences as shown in SEQ ID NO: 5 or SEQ ID NO: 6. The binding domain of ILT2 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the binding domain of ILT4 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4. Either of the two synthetic polypeptides is used as the extracellular region of the chimeric antigen receptor. A signal peptide sequence derived from CD8α is linked to its N-terminus, and a hinge region sequence derived from CD8α, a transmembrane domain sequence derived from CD8α, a 4-1BB sequence, and a CD3ζ sequence are sequentially linked to its C-terminus. Its amino acid sequence is shown in SEQ ID NO:7-11, and its nucleotide sequence is shown in SEQ ID NO:12-16. The two chimeric receptors that are ultimately formed contain ILT2-D1D2+Linker+ILT4-D1D2 and ILT4-D1D2+Linker+ILT2-D1D2, respectively, and are named CAR1 and CAR2.

[0114] Without substantially affecting the domain activity (retaining at least 95% activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the domain or its functional fragments. These are all considered to be included within the scope of protection of this invention. The variant sequences described in this invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0115] Polynucleotides, expression vectors, recombinant viruses, cells, compositions

[0116] The present invention also provides an isolated nucleic acid encoding a CAR, an expression vector containing the nucleic acid, and a recombinant virus. The nucleic acid molecule encodes the aforementioned CAR, and the nucleic acid is preferably an expression cassette obtained by genetic engineering methods.

[0117] Expression vectors can refer to cloning vectors or recombinant vectors. They can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, and pUC57.

[0118] According to embodiments of the present invention, enhanced immune cells can be obtained using any of the following methods or combinations thereof:

[0119] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0120] 2) Integrate one or more circular or linear exogenous DNA fragments into target cells at specific sites. Site-specific integration can improve the efficiency of homologous recombination of exogenous DNA by introducing endonucleases at the target site; it can also improve the efficiency of homologous recombination by other physical means, such as electroporation.

[0121] 3) Introduce one or more double-strand breaks into target cells at specific sites using one or more endonucleases that can recognize specific sites. The target cells then generate base insertions or deletions at the specific sites by joining non-homologous ends.

[0122] 4) Introduce one or more double-strand breaks into target cells at specific sites using one or more endonucleases that can recognize specific sites, while providing exogenous circular or linear DNA fragments. The cells then generate base insertions or deletions at selected sites through homologous recombination.

[0123] The target cells at this point can be the immune cells, stem cells, or progenitor cells mentioned above, or donor cells used for reprogramming into iPSCs, or any derived cells from stem cells or progenitor cells during their differentiation into immune cells. The exogenous DNA fragment may contain a gene expression frame, i.e., at least components such as a promoter and an open reading frame; or it may not contain a gene expression frame.

[0124] In some preferred embodiments, the nucleic acid molecules are species-optimized to be more easily expressed in mammalian cells.

[0125] This invention also provides an expression vector comprising the isolated nucleic acid molecules described above. When ligating the isolated polynucleotides to the vector, the polynucleotides can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotides. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotides and control elements need to be operatively linked. In this context, "operatively linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides used to encode different domains of the CAR can be independently inserted into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, etc.

[0126] This application provides a recombinant virus that can express a specific chimeric antigen receptor targeting HLA-G and can infect immune response cells.

[0127] This invention also provides a recombinant cell containing the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, which are then used to express the chimeric antigen receptor provided by this invention. The recombinant cell is derived from immune response cells, including at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells. The immune cells are obtained from peripheral blood, umbilical cord blood, or tissues and organs, or are differentiated from stem cells or immune cell progenitor cells; the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells that have not undergone in vivo development within 14 days of fertilization.

[0128] The compositions provided by this invention contain at least one of the chimeric antigen receptor, polynucleotide, expression vector, recombinant virus, or recombinant cell as described above. Further, the compositions contain a pharmaceutically acceptable carrier. In some embodiments, the compositions comprise combinations that are separate in time and / or space, provided they can work together to achieve the objectives of this invention. For example, the components contained in the composition may be administered to a subject as a whole or separately. When the components contained in the composition are administered to a subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0129] In some embodiments, the drug carrier comprises any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, flow aid or lubricant, etc., suitable for a specific target dosage form. The use of any conventional excipients, except those incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this invention.

[0130] In one specific embodiment of the present invention, suitable target cell lines are screened for testing the killing ability of newly produced CAR-NK cells. The screened target cells can be used for both in vitro experiments and in vivo experiments in mice.

[0131] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0132] Table 1 shows the nucleotide and amino acid sequences involved in this invention. Based on the sequences provided by this invention, those skilled in the art can easily obtain the CAR-NK cells provided by this invention.

[0133] Table 1

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] Example 1: Design of a chimeric antigen receptor that specifically targets and binds to human HLA-G

[0143] The inventors designed two synthetic peptides that can target and bind to human HLA-G. Both synthetic peptides are composed of peptide sequences derived from ILT2 and ILT4. The synthetic peptides derived from ILT2 and ILT4 have amino acid sequences as shown in SEQ ID NO: 5 or SEQ ID NO: 6. The binding domain of ILT2 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the binding domain of ILT4 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4. Either of the two synthetic peptides is used as the extracellular region of the chimeric antigen receptor. A signal peptide sequence derived from CD8α is attached to its N-terminus, and a hinge region sequence derived from CD8α, a transmembrane domain sequence derived from CD8α, a 4-1BB sequence, and a CD3ζ sequence are sequentially attached to its C-terminus. Its amino acid sequence is shown in SEQ ID NO:7-11, and its nucleotide sequence is shown in SEQ ID NO:12-16. The two chimeric receptors that are ultimately formed contain ILT2-D1D2+Linker+ILT4-D1D2 and ILT4-D1D2+Linker+ILT2-D1D2, respectively, and are named CAR1 and CAR2.

[0144] Example 2: Construction of an expression plasmid vector containing a chimeric antigen receptor that specifically targets and binds to human HLA-G

[0145] The construction of expression plasmid vectors (pCDH-CAR1 and pCDH-CAR2) containing specific chimeric antigen receptors that target and bind to human HLA-G specifically includes the following steps:

[0146] (1) Genewiz synthesized DNA sequences targeting specific chimeric antigen receptors (CAR1 and CAR2) that bind to human HLA-G into the pUC57 vector. The DNA sequences are shown in SEQ ID NO:19-20, and the corresponding encoded amino acid sequences are shown in SEQ ID NO:21-22.

[0147] (2) The pCDH-EV vector was double-digested with EocR1 and BamH1 endonucleases. After electrophoresis on a 1% agarose gel for 20 min, the double-digested vector was recovered by cutting the gel.

[0148] (3) A nucleotide fragment of a chimeric antigen receptor specifically targeting human HLA-G was obtained by PCR from a pUC57 vector containing a DNA sequence of a chimeric antigen receptor specifically targeting human HLA-G synthesized in (1), using primer pair 1, wherein the nucleotide sequences of the primer pair (primer 1F, primer 1R) are as shown in SEQ ID NO:23-24.

[0149] (4) Homologously recombine the double-digested vector obtained in (2) and (3) with the nucleotide fragment that specifically targets and binds to the chimeric antigen receptor of human HLA-G;

[0150] (5) The constructed expression plasmid vectors containing chimeric antigen receptors that specifically target and bind to human HLA-G (pCDH-CAR1, pCDH-CAR2) were sequenced for verification. The sequencing results confirmed that the construction was successful.

[0151] Example 3: Construction and identification of cell lines containing chimeric antigen receptors that specifically target and bind to human HLA-G

[0152] Expression plasmids (pCDH-CAR1 and pCDH-CAR2), pMD2G, and psPAX2 were co-transfected into 15cm culture dishes containing 90% HEK293-T cells. After three days, the cell supernatant was collected, and the viral solution was filtered through a 0.45μm filter and the virus was concentrated to obtain lentiviruses of expression plasmids (pCDH-CAR1 and pCDH-CAR2).

[0153] NK92 cells were added to a final concentration of 1000 U / mL human recombinant IL-2 and 20 ng / mL human IL-21 and cultured for 24 hours. Then, the lentivirus and the infection-promoting reagent polybrene were added at an MOI of 1, and the cells were infected for 24 hours. Afterward, an appropriate amount of culture medium was added, and the infection efficiency of NK92 cells was detected by flow cytometry after 48 hours. The specific procedures are as follows.

[0154] (1) Blank control group: NK cells that were not infected with viral fluid;

[0155] (2) Experimental group: 2 NK cells infected with different CAR1 or CAR2 viruses respectively.

[0156] The experimental and control groups were washed twice with PBS and resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA). Following the antibody instructions, PE-labeled anti-human ILT2 antibody and APC-labeled anti-human ILT4 antibody were added to the cell suspensions of the test and control groups, and incubated at 4°C for 30 minutes. Stained cells were obtained using flow cytometry, and the results were analyzed using FlowJo software. The flow cytometry results are shown below. Figure 1 As shown, CAR molecule expression was almost undetectable in the control group, while the expression rate of CAR molecules in each experimental group reached over 90%. Figure 1 The flow cytometry results show that the cells collected in this embodiment express a specific chimeric antigen receptor targeting HLA-G.

[0157] Example 4: Screening of target cell lines

[0158] To screen suitable target cell lines for testing the killing ability of newly produced CAR-NK cells, the inventors analyzed the expression of HLA-G in tumor cell lines such as ovarian cancer SKOV3, colon cancer NCI-H716, and leukemia cells K562. The tested tumor cells were collected, washed twice with PBS, and resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA). Cell counts were performed, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / cells. 6 / ml; add PE-HLA-G and isotype control antibodies respectively, incubate at 4℃ for 30 minutes; wash twice with PBS and resuspend in FACS solution, analyze cell fluorescence by flow cytometry, and analyze the results using FlowJo software. Figure 2 As shown, K562, NCI-H716, and SKOV3 cells in the tested tumor cell group all showed high expression of HLA-G antigen, while the liver cancer cell line Huh7 did not express HLA-G.

[0159] Example 5: In vitro killing effect of chimeric antigen receptor cells that specifically target and bind to human HLA-G on target cells.

[0160] Next, the in vitro killing activity of CAR1-NK and CAR2-NK cells was tested using PI and CFSE staining. Specifically, for the tumor cell lines K562, NCI-H716, and Huh7, after CSFE fluorescence staining, the three target cell lines were sampled at 2 × 10⁻⁶ cells per well. 4The cells were seeded at a concentration of [number] cells / ml in culture plates. Two experimental groups and one control group were set up for each target cell line. The experimental groups contained a suspension of HLA-G-targeting CAR-NK cells obtained in Example 3; the blank control group contained NK cells infected with an empty vector virus. In the experimental groups, the HLA-G-targeting CAR-NK cells from Example 3 were mixed with the target cells at a 1:1 effector-to-target ratio.

[0161] For the SKOV3 tumor cell line, after adding 5×10 3 After the target cells adhered to the E-plate, experimental and control group cells were added, and the growth of the target cells was then detected using an RTCA instrument.

[0162] Similarly, in the blank control group, NK cells and target cells were mixed at a 1:1 effector-to-target ratio. After culturing for 4 hours, the cells were centrifuged to remove the supernatant, washed, stained with PI, and stained cells were obtained by flow cytometry. The results were analyzed using FlowJo software. Figure 3 The results shown are the tumor cell killing rate test results using K562 leukemia cells as the target cells. Figure 3 It can be seen that the HLA-G-targeting specific CAR-NK cells in Example 3 have a significant killing effect on K562 leukemia cells (significantly higher than the control group). Figure 4 The results shown are the tumor cell killing rate test results using NCI-H716 colon cancer cells as target cells. Figure 4 As can be seen, the HLA-G-targeting specific CAR-NK cells in Example 3 have a significant killing effect on NCI-H716 colon cancer cells (significantly higher than the control group). Figure 5 The results show the tumor cell killing rate test results using SKOV3 ovarian cancer cells as target cells. Figure 5 It can be seen that the HLA-G-targeting specific CAR-NK cells in Example 3 have a significant killing effect on SKOV3 ovarian cancer (significantly higher than the control group). From Figure 6 It can be seen that the HLA-G-targeting specific CAR-NK cells in Example 3 did not significantly enhance the killing effect on HLA-G-negative Huh7 cells (the killing effect was similar to that of the control group).

[0163] Example 6: In vivo experimental verification of the effectiveness of chimeric antigen receptor NK cells that target and bind to human HLA-G in killing target cells.

[0164] Take 2×10 6K562-luciferase cells were injected intravenously into 5-week-old NCG mice. Five days after tumor formation, fluorescein was injected intraperitoneally into the mice. The K562 tumors in the NCG mice were imaged using a small animal in vivo imaging system. Mice with similar tumor formation were randomly divided into three groups: an experimental group and two control groups (one untreated control and one treated with standard NK92). Subsequently, 1×10⁶ K562-luciferase cells were injected intravenously into the experimental group mice. 7 CAR2-NK92 cells were injected intravenously into the tail vein of a control group of mice at a dose of 1×10⁻⁶. 7 Ordinary, unmodified NK92 cells were used. Treatment was then administered weekly, along with small animal tumor imaging. During this period, IL-2 was injected intraperitoneally every 3 days, with each animal receiving 5 × 10⁶ cells per injection. 4 U, a total of 3 treatments were given. Figure 7 The image shows the killing rate of CAR2-NK cells against K562 leukemia cells after tumor bearing. From... Figure 7 It can be seen that HLA-G-targeting specific CAR2-NK cells have a significant killing effect on K562 leukemia cells in vivo (significantly higher than the control group).

[0165] Take 2×10 6 One SKOV3 cell was subcutaneously injected into 5-week-old NCG mice. Seven days after tumor formation, tumor size was measured using calipers. Mice with similar tumor development were randomly divided into three groups: an experimental group and two control groups (one untreated control and one treated with standard NK92). Subsequently, 1×10⁻⁶ SKOV3 cells were injected intravenously into the tail vein of the experimental group mice. 7 CAR2-NK92 cells were injected intravenously into the tail vein of a control group of mice at a dose of 1×10⁻⁶. 7 Ordinary, unmodified NK92 cells were used. Treatment was then administered weekly, with subcutaneous tumor size measured during the treatment. IL-2 was injected intraperitoneally every 3 days, with each cell receiving 5 × 10⁻⁶ cells. 4 U, a total of 3 treatments were given. Figure 8 The image shows the therapeutic effect of CAR2-NK cells on SKOV3 ovarian cancer cells after tumor bearing. From... Figure 8 It can be seen that HLA-G-targeting specific CAR-NK cells have a significant tumor-suppressing effect on SKOV3 ovarian cancer cells in vivo (significantly higher than the control group).

[0166] Take 1×10 7NCI-H716 cells were subcutaneously injected into 5-week-old NCG mice. Ten days after tumor formation, tumor size was measured using calipers. Mice with similar tumor development were randomly divided into three groups: an experimental group and two control groups (one untreated control and one treated with standard NK92). Subsequently, 1×10⁶ NCI-H716 cells were injected intravenously into the tail vein of the experimental group mice. 7 CAR2-NK92 cells were injected intravenously into the tail vein of a control group of mice at a dose of 1×10⁻⁶. 7 Ordinary, unmodified NK92 cells were used. Treatment was then administered weekly, with subcutaneous tumor size measurements taken during the treatment period. IL-2 was injected intraperitoneally every 3 days, with each cell receiving 5 × 10⁻⁶ cells. 4 U, a total of 3 treatments were given. Figure 9 The image shows the killing rate of CAR2-NK cells against NCI-H716 colon cancer cells after tumor bearing. From... Figure 9 It can be seen that the HLA-G-targeting specific CAR-NK cells of the present invention have a significant tumor-suppressing effect on NCI-H716 colon cancer cells in vivo (significantly higher than the control group).

[0167] The results above show that the CAR-NK cells provided by this invention have the ability to specifically recognize and kill HLA-G positive tumor cells, both in vitro and in vivo.

[0168] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0169] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0170] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor that specifically targets and binds to HLA-G, characterized in that, The chimeric antigen receptor includes: The extracellular domain includes at least one binding domain for ILT2 targeting HLA-G and at least one binding domain for ILT4 targeting HLA-G. The extracellular domain further includes a signal peptide linked to the binding domains for ILT2 and ILT4 targeting HLA-G. The extracellular domain is connected to a transmembrane domain via a hinge domain derived from the CD8α hinge region. A transmembrane domain, which is connected to the extracellular domain, and the transmembrane domain originates from the CD8α transmembrane structural region; An intracellular domain, which is connected to the transmembrane domain, the intracellular domain including an immune receptor tyrosine activation motif and a co-stimulatory signaling domain, the co-stimulatory signaling domain and the immune receptor tyrosine activation motif being connected by a linker peptide. The immune receptor tyrosine activation motif originates from the intracellular signal transduction domain of the CD3ζ chain; The co-stimulatory signal domain originates from the intracellular signal transduction domain of 4-1BB cells; The binding domain of ILT2 that targets and binds to HLA-G has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2. The binding domain of ILT4 that targets HLA-G has an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4; The binding domain of ILT2 that targets HLA-G and the binding domain of ILT4 that targets HLA-G are connected by a linker peptide. in, The amino acid sequences of the synthetic polypeptides derived from ILT2 and ILT4 are shown in SEQ ID NO: 5 or SEQ ID NO: 6; The extracellular domain of the chimeric antigen receptor is constructed using either the synthetic polypeptide derived from ILT2 or ILT4. A signal peptide sequence derived from CD8 is attached to the N-terminus of the extracellular domain of the chimeric antigen receptor, and a hinge region sequence derived from CD8, a transmembrane domain sequence derived from CD8, a 4-1BB sequence, and a CD3ζ sequence are sequentially attached to the C-terminus of the extracellular domain of the chimeric antigen receptor. The chimeric antigen receptor comprises either ILT2-D1D2+Linker+ILT4-D1D2 or ILT4-D1D2+Linker+ILT2-D1D2.

2. The chimeric antigen receptor according to claim 1, characterized in that, The signal peptide has the amino acid sequence shown in SEQ ID NO:

7.

3. The chimeric antigen receptor according to claim 1, characterized in that, The hinge domain has an amino acid sequence as shown in SEQ ID NO:

8.

4. The chimeric antigen receptor according to claim 1, characterized in that, The transmembrane domain has an amino acid sequence as shown in SEQ ID NO:

9.

5. The chimeric antigen receptor according to claim 1, characterized in that, The immune receptor tyrosine activation motif has an amino acid sequence as shown in SEQ ID NO:

11.

6. The chimeric antigen receptor according to claim 1, characterized in that, The co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:

10.

7. The chimeric antigen receptor according to claim 1, characterized in that, The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO:

22.

8. An isolated polynucleotide, characterized in that, The polynucleotide encodes the chimeric antigen receptor according to any one of claims 1-7.

9. An expression carrier, characterized in that, The expression vector carries the polynucleotide of claim 8.

10. A recombinant virus, characterized in that, The recombinant virus carries the polynucleotide of claim 8 or the expression vector of claim 9, and the recombinant virus is capable of expressing the chimeric antigen receptor of any one of claims 1-7.

11. A recombinant cell, characterized in that, The recombinant cells carry the polynucleotide of claim 8 or the expression vector of claim 9 or are infected with the recombinant virus of claim 10, and the recombinant cells are capable of expressing the chimeric antigen receptor of any one of claims 1-7.

12. The recombinant cell according to claim 11, characterized in that, The recombinant cells are derived from immune cells.

13. The recombinant cell according to claim 12, characterized in that, The immune cells are obtained through isolation from peripheral blood, umbilical cord blood, or tissues and organs, or the immune cells are obtained by differentiation from stem cells or immune cell progenitor cells.

14. The recombinant cell according to claim 13, characterized in that, The stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells that have not undergone in vivo development and are within 14 days of fertilization.

15. The recombinant cell according to claim 12, characterized in that, The immune cells include at least one selected from T cells, B cells, monocytes, and NK cells.

16. The recombinant cell according to claim 15, characterized in that, The monocytes are selected from dendritic cells and macrophages.

17. The recombinant cell according to claim 15, characterized in that, The T cells are selected from cytotoxic T cells, helper T cells, NKT cells, regulatory T cells, and γδT cells.

18. A composition, characterized in that, Contains at least one of the following: The chimeric antigen receptor according to any one of claims 1-7; The polynucleotide of claim 8; The expression vector according to claim 9; The recombinant virus according to claim 10; The recombinant cells according to any one of claims 11-17.

19. Use of the composition of claim 18 in the preparation of a medicament for at least one of leukemia, ovarian cancer, and colon cancer.

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